Down-regulation of acyl-CoA oxidase gene expression and increased NF-κB activity in etomoxir-induced cardiac hypertrophy
2003; Elsevier BV; Volume: 44; Issue: 2 Linguagem: Inglês
10.1194/jlr.m200294-jlr200
ISSN1539-7262
AutoresÀgatha Cabrero, Manuel Merlos, Juan C. Laguna, Manuel Vázquez‐Carrera,
Tópico(s)Fuel Cells and Related Materials
ResumoActivation of nuclear factor-κB (NF-κB) is required for hypertrophic growth of cardiomyocytes. Etomoxir is an irreversible inhibitor of carnitine palmitoyltransferase I (CPT-I) that activates peroxisome proliferator-activated receptor α (PPARα) and induces cardiac hypertrophy through an unknown mechanism. We studied the mRNA expression of genes involved in fatty acid oxidation in the heart of mice treated for 1 or 10 days with etomoxir (100 mg/kg/day). Etomoxir administration for 1 day significantly increased (4.4-fold induction) the mRNA expression of acyl-CoA oxidase (ACO), which catalyzes the rate-limiting step in peroxisomal β-oxidation. In contrast, etomoxir treatment for 10 days dramatically decreased ACO mRNA levels by 96%. The reduction in ACO expression in the hearts of 10-day etomoxir-treated mice was accompanied by an increase in the mRNA expression of the antioxidant enzyme glutathione peroxidase and the cardiac marker of oxidative stress bax. Moreover, the activity of the redox-regulated transcription factor NF-κB was increased in heart after 10 days of etomoxir treatment.Overall, the findings here presented show that etomoxir treatment may induce cardiac hypertrophy via increased cellular oxidative stress and NF-κB activation. Activation of nuclear factor-κB (NF-κB) is required for hypertrophic growth of cardiomyocytes. Etomoxir is an irreversible inhibitor of carnitine palmitoyltransferase I (CPT-I) that activates peroxisome proliferator-activated receptor α (PPARα) and induces cardiac hypertrophy through an unknown mechanism. We studied the mRNA expression of genes involved in fatty acid oxidation in the heart of mice treated for 1 or 10 days with etomoxir (100 mg/kg/day). Etomoxir administration for 1 day significantly increased (4.4-fold induction) the mRNA expression of acyl-CoA oxidase (ACO), which catalyzes the rate-limiting step in peroxisomal β-oxidation. In contrast, etomoxir treatment for 10 days dramatically decreased ACO mRNA levels by 96%. The reduction in ACO expression in the hearts of 10-day etomoxir-treated mice was accompanied by an increase in the mRNA expression of the antioxidant enzyme glutathione peroxidase and the cardiac marker of oxidative stress bax. Moreover, the activity of the redox-regulated transcription factor NF-κB was increased in heart after 10 days of etomoxir treatment. Overall, the findings here presented show that etomoxir treatment may induce cardiac hypertrophy via increased cellular oxidative stress and NF-κB activation. Energy demand of the heart depends on the oxidation of a variety of substrates, mainly fatty acids and glucose. This process is regulated during development and under various physiological and pathophysiological conditions depending on the substrate availability (1Van Bilsen M. J. van der Vusse G. Reneman R.S. Transcriptional regulation of metabolic processes: implications for cardiac metabolism.Pflügers Arch. 1998; 437: 2-14Google Scholar, 2Stanley W.C. Lopaschuk G.D. McCormack J.G. Regulation of energy substrate metabolism in the diabetic heart.Cardiovasc. Res. 1997; 34: 25-33Google Scholar). Thus, during the fetal period, cardiac metabolism relies on glucose, whereas after birth, myocardial energy increasingly switches from glucose to fatty acids oxidation, the latter being the major source of energy in the adult mammalian heart. In addition, during the development of cardiac hypertrophy in rodents and humans, a dramatic reduction in fatty acid oxidation is detected, since a shift in the source of energy is observed from fatty acids to glucose (1Van Bilsen M. J. van der Vusse G. Reneman R.S. Transcriptional regulation of metabolic processes: implications for cardiac metabolism.Pflügers Arch. 1998; 437: 2-14Google Scholar). The adjustments of cardiac metabolism to the substrate availability seem to involve changes in the transcriptional control of genes implicated in the transport and metabolism of fatty acids and glucose, which in turn are under the control of a class of transcription factors called peroxisome proliferator-activated receptors (PPARs). Three different PPAR subtypes (α, δ/β, and γ) have been identified to date. PPARα is expressed primarily in tissues with a high level of fatty acid catabolism, such as liver, brown fat, kidney, heart, and skeletal muscle (3Braissant O. Foufelle F. Scotto C. Dauça M. Wahli W. Differential expression of peroxisome proliferator-activated receptors (PPARs): tissue distribution of PPAR-alpha, -beta, and -gamma in the adult rat.Endocrinology. 1996; 137: 354-366Google Scholar, 4Desvergne B. Wahli W. Peroxisome proliferator-activated receptors: nuclear control of metabolism.Endocr. Rev. 1999; 20: 649-688Google Scholar). PPARβ is ubiquitously expressed, and PPARγ has a restricted pattern of expression, mainly in white and brown adipose tissues, whereas other tissues such as skeletal muscle and heart contain limited amounts (3Braissant O. Foufelle F. Scotto C. Dauça M. Wahli W. Differential expression of peroxisome proliferator-activated receptors (PPARs): tissue distribution of PPAR-alpha, -beta, and -gamma in the adult rat.Endocrinology. 1996; 137: 354-366Google Scholar). PPARs are activated by ligands, such as naturally occurring fatty acids, which are activators of all three PPAR subtypes (5Kliewer S.A. Sundseth S.A. Jones S.A. Brown P.J. Wisely G.B. Koble C.S. Devchand P. Wahli W. Willson T.M. Lenhard J.M. Lehmann J.M. Fatty acids and eicosanoids regulate gene expression through direct interactions with peroxisome proliferator-activated receptors alpha and gamma.Proc. Natl. Acad. Sci. USA. 1997; 94: 4318-4328Google Scholar, 6Krey G. Braissant O. L'Horset F. Kalkhoven E. Perroud M. Parker M.G. Wahli W. Fatty acids, eicosanoids, and hypolipidemic agents identified as ligands of peroxisome proliferator-activated receptors by coactivator-dependent receptor ligand assay.Mol. Endocrinol. 1997; 11: 779-791Google Scholar). In addition to fatty acids, several synthetic compounds, such as fibrates and thiazolidinediones, bind and activate specific PPAR subtypes. In order to be transcriptionaly active, PPARs need to heterodimerize with the retinoid X receptor (RXR). PPAR-RXR heterodimers bind to DNA-specific sequences called peroxisome proliferator response elements (PPREs), consisting of an imperfect direct repeat of the consensus binding site for nuclear hormone receptors (AGGTCA) separated by one nucleotide (DR-1). This direct repeat is known to bind potential competitors such as homodimers of other nuclear receptors including RXRα, chicken ovalbumin upstream promoter transcription factor II (COUP-TF-II, also called apolipoprotein A-I regulatory protein, ARP-1), and hepatic nuclear factor-4 (HNF-4) (4Desvergne B. Wahli W. Peroxisome proliferator-activated receptors: nuclear control of metabolism.Endocr. Rev. 1999; 20: 649-688Google Scholar). It has been previously reported that development of pressure overload-induced ventricular hypertrophy in mice, which involves a shift in the substrate utilization from fatty acids to glucose, is associated with deactivation of PPARα (7Sack M.N. Disch D.L. Rockman H.A. Kelly D.P. A role for Sp and nuclear receptor transcription factors in a cardiac hypertrophy growth program.Proc. Natl. Acad. Sci. USA. 1997; 94: 6438-6443Google Scholar, 8Barger P.M. Brandt J.M. Leone T.C. Weinheimer C.J. Kelly D.P. Deactivation of peroxisome proliferator-activated receptor-a during cardiac hypertrophic growth.J. Clin. Invest. 2000; 105: 1723-1730Google Scholar). Further, it has been shown that the energy substrate switch observed in cardiac hyperthrophy involves reactivation of fetal transcriptional control via members of the Sp and COUP-TF families of transcription factors (7Sack M.N. Disch D.L. Rockman H.A. Kelly D.P. A role for Sp and nuclear receptor transcription factors in a cardiac hypertrophy growth program.Proc. Natl. Acad. Sci. USA. 1997; 94: 6438-6443Google Scholar). These changes may account for the down-regulation of enzymes involved in fatty acid oxidation. However, little is known about the effects of several drugs leading to cardiac hyperthrophy. In fact, several studies have shown that inhibition of mitochondrial β-oxidation by carnitine palmitoyltransferase I (CPT-I) inhibitors, which were developed for the treatment of type 2 diabetes mellitus, is sufficient to cause cardiac hypertrophy (9Vetter R. Rupp H. CPT-1 inhibition by etomoxir has a chamber-related action on cardiac sarcoplasmic reticulum and isomyosins.Am. J. Physiol. 1994; 267: H2021-H2099Google Scholar). In order to delineate the molecular mechanisms involved in etomoxir-induced cardiac hypertrophy, we treated mice with this irreversible inhibitor of CPT-I. Treatment for 1 day with this drug, which in addition activates PPARα (10Forman B.A. Chen J. Evans R.M. 15-Deoxy-delta 12, 14-prostaglandin J2 is a ligand for the adipocyte determination factor PPARgamma.Proc. Natl. Acad. Sci. USA. 1997; 94: 4312-4317Google Scholar), resulted in a significant increase in the mRNA expression of acyl-CoA oxidase (ACO), the gene that catalyzes the rate-limiting step in peroxisomal β-oxidation. In contrast, a dramatic reduction in ACO mRNA levels was observed after etomoxir administration for 10 days. On the other hand, the mRNA levels of PPARα, and of several of its target genes involved in mitochondrial β-oxidation, were not significantly modified by etomoxir after 10 days of treatment. The fall in ACO expression after 10 days of etomoxir treatment was accompanied by increased activity of the redox-regulated transcription factor, nuclear factor κB (NF-κB). Overall, the results presented here suggest that etomoxir increases oxidative stress in cardiomyocytes, leading to NF-κB activation, which is required for the hypertrophic growth of cardiomyocytes (11Purcell H.N. Tang G. Yu C. Mercurio F. DiDonato J.A. Lin A. Activation of NF-kappa B is required for hypertrophic growth of primary rat neonatal ventricular cardiomyocytes.Proc. Natl. Acad. Sci. USA. 2001; 98: 6668-6673Google Scholar). The negative correlation in heart between enhanced oxidative stress and the reduction ACO expression suggests that peroxisomal β-oxidation may be involved in cardiac hypertrophy after etomoxir treatment. Twenty male Swiss mice from Harlan (Barcelona, Spain) were used. They were maintained under standard conditions of illumination (12-h light/dark cycle) and temperature (21 ± 1°C) and fed a standard diet. The mice were randomly distributed into two groups. Each group was administered, respectively, either 0.5% carboximethyl cellulose (control group) or 100 mg/kg/day of etomoxir (dissolved in 0.5% carboximethyl cellulose) per os once a day for either 1 or 10 days (1 ml/100 g of body weight). Food and water were given ad libitum. Twenty-four hours after the last administration, mice were killed under pentobarbitone anesthesia to collect blood samples and to isolate hearts. Blood samples, obtained by cardiac puncture, were collected in EDTA tubes, and plasma was obtained by centrifugation at 2,200 g for 10 min at 4°C. Plasma glucose (Roche, Barcelona, Spain), triglycerides (Sigma), and nonesterified fatty acids (Wako, Germany) levels were determined with colorimetric test. Hearts were rapidly removed, washed in ice-cold 0.9% NaCl, frozen in liquid nitrogen, and stored at −80°C. Animal handling and disposal were performed in accordance with law 5/1995, 21st July, of the Generalitat de Catalunya. Total RNA was isolated by using the Ultraspec reagent (Biotecx, Houston). Relative levels of specific mRNAs were assessed by the reverse transcription-polymerase chain reaction (RT-PCR). cDNA was synthesized from RNA samples by mixing 0.5 μg of total RNA, 125 ng of random hexamers as primers in the presence of 50 mM Tris-HCl buffer (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol, 200 units of Moloney murine leukemia virus reverse transcriptase (Life Technologies), 20 units of RNAsin (Life Technologies), and 0.5 mM of each dNTP (Sigma) in a total volume of 20 μl. Samples were incubated at 37°C for 60 min. A 5 μl aliquot of the RT reaction was then used for subsequent PCR amplification with specific primers. Each 25-μl PCR reaction contained 5 μl of the RT reaction, 1.2 mM MgCl2, 200 μM dNTPs, 1.25 μCi [32P]dATP (3,000 Ci/mmol, Amersham), 1 unit of Taq polymerase (Ecogen, Barcelona, Spain), 0.5 μg of each primer, and 20 mM Tris-HCl, pH 8.5. To avoid unspecific annealing, cDNA and Taq polymerase were separated from primers and dNTPs by using a layer of paraffin (reaction components contact only when paraffin fuses, at 60°C). The sequences of the sense and antisense primers used for amplification were: PPARα, 5′-GGCTCGGAGGGCTCTGTCATC-3′ and 5′-ACATGCACTGGCAGCAGTGGA-3′; M-CPT-I, 5′-TTCACTGTGACCCCAGACGGG-3′ and 5′-AATGGACCAGCCCCATGGAGA; MCAD, 5′-TCGAAAGCGGCTCACAAGCAG-3′ and 5′-CACCGCAGCTTTCCGGAATGT-3′; UCP-3, 5′-GGAGCCATGGCAGTGACCTGT-3′ and 5′-TGTGATGTTGGGCCAAGTCCC-3′; UCP-2, 5′-AACAGTTCTACACCAAGGGC-3′ and 5′-AGCATGGTAAGGGCACAGTG-3′; ACO, 5′-ACTATATTTGGCCAATTTTGTG-3′ and 5′-TGTGGCAGTGGTTTCCAAGCC-3′; CTE, 5′-CAGCCACCCCGAGGTAAAAGG-3′ and 5′-CCTTGAGGCCATCCTTGGTCA-3′; RXRα, 5′-GCTCTCCAACGGGTCGAGGCT-3′ and 5′-TGGGTGTGGTGGGTACCGACA-3′; RXRγ, 5′-CCATGAAGACATGCCGGTGGA-5′ and 5′-CCCGGTGCAGAATGACCTGGT-3′; PGC-1 5′-CCCGTGGATGAAGACGGATTG-3′ and 5′-GTGGGTGTGGTTTGCTGCATG-3′; GPX, 5′-CGGCACAGTCCACCGTGTATG-3′ and 5′-ACTGATTGCACGGGAAACCGA-3′; BAX, 5′-GGCCCACCAGCTCTGAACAGA-3′ and 5′-AGCTGCCACCCGGAAGAAGAC-3′; and adenosyl phosphoribosyl transferase (APRT), 5′-AGCTTCCCGGACTTCCCCATC-3′ and 5′-GACCACTTTCTGCCCCGGTTC-3′. PCR was performed in an MJ Research Thermocycler equipped with a peltier system and temperature probe. After an initial denaturation for 1 min at 94°C, PCR was performed for 20 (MCAD), 22 (GPX), 23 (UCP-2, PGC-1), 25 (UCP-3, PPARα, BAX), 26 (RXRα and RXRγ), 27 (CTE), and 28 (M-CPT-I, ACO) cycles. Each cycle consisted of denaturation at 92°C for 1 min, primer annealing at 60°C (except 58°C for ACO), and primer extension at 72°C for 1 min and 50 s. A final 5-min extension step at 72°C was performed. Five microliters of each PCR sample was electrophoresed on a 1 mm thick 5% polyacrylamide gel. The gels were dried and subjected to autoradiography using Kodak X-ray films to show the amplified DNA products. Amplification of each gene yielded a single band of the expected size (PPARα: 645 bp; M-CPT-I: 222 bp; MCAD: 216 bp; UCP-3: 179 bp; UCP-2: 471 bp; ACO: 195 bp; CTE: 224 bp; RXRα: 202 bp; RXRγ: 220 bp; PGC-1: 228 bp; GPX: 210 bp; BAX: 256 bp; and APRT: 329 bp). Preliminary experiments were carried out with varying amounts of cDNA to determine nonsaturating conditions of PCR amplification for all the genes studied. Therefore, under these conditions, relative quantification of mRNA was assessed by the RT-PCR method used in this study (12Freeman W.M. Walker S.J. Vrana E.V. Quantitative RT-PCR: pitfalls and potential.Biotechniques. 1999; 26: 112-125Google Scholar). Radioactive bands were quantified by videodensitometric scanning (Vilbert Lourmat Imaging). The results for the expression of specific mRNAs are always presented relative to the expression of the control gene (aprt). Crude nuclear extracts were isolated using the Dignam method (13Dignam J.D. Lebovitz R.M. Roeder R.G. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei.Nucleic Acids Res. 1983; 11: 1475-1489Google Scholar) with the modifications described by Helenius et al. (14Helenius M. Hänninen M. Lehtinen S. Salminen A. Aging-induced up-regulation of nuclear binding activities of oxidative stress responsive NF-kB transcription factor in mouse cardiac muscle.J. Mol. Cell. Cardiol. 1996; 28: 487-498Google Scholar). Frozen hearts were weighed, transferred to Corning tubes, and ice-cold hypotonic buffer (1.5 mM MgCl2, 10 mM KCl, 0.2 mM PMSF, 0.5 mM DTT, 10 mM HEPES, pH 7.9) was added to each sample. The volume was proportional to the weight of the tissue so as to give 15% homogenates. The tissues were left to thaw in an ice bath and homogenized (2 × 5 s) using a Polytron (Kinematica, Germany). Homogenates were incubated for 10 min on ice and centrifuged (25,000 g, 15 min, 4°C). Pellets were washed once with the same volume of hypotonic buffer used in the homogenization step and centrifuged (10,000 g, 4°C, 15 min). Supernatants were discarded and pellets were suspended in ice-cold low salt buffer (25% glycerol, 1.5 mM MgCl2, 0.2 mM EDTA, 0.2 mM PMSF, 0.5 mM DTT, 20 mM KCl, 20 mM Hepes, pH 7.9) using half of the volume of the hypotonic buffer. Nuclear proteins were released by adding high-salt buffer (25% glycerol, 1.5 mM MgCl2, 0.2 mM EDTA, 0.2 mM PMSF, 0.5 mM DTT, 1.2 M KCl, 20 mM Hepes, pH 7.9) drop-by-drop using half of the volume of the low-salt buffer. Samples were incubated on ice for 30 min. During incubation, the tubes were smoothly mixed frequently. Samples were centrifuged (25,000 g, 30 min, 4°C), and supernatants were collected in microfuge tubes and stored in aliquots at −80°C. The protein concentration of the nuclear extracts was then measured. Electrophoretic mobility shift assays (EMSAs) were performed using double-stranded oligonucleotides (Santa Cruz Biotechnology) for the consensus binding sites of PPRE (5′-CAAAACTAGGTCAAAGGTCA-3′), mutant PPRE (5′-CAAAACTAGCACAAAGCACA-3′), Sp1 (5′-ATTCGATCGGGGCGGGGCGAGC-3′), and Oct-1 (5′-TGTCGAATGCAAATCACTAGAA-3′). The NF-κB consensus oligonucleotide (5′AGTTGAGGGGACTTTCCCAGGC-3′) was from Promega. Oligonucleotides were labeled in the following reaction: 1 μl of oligonucleotide (20 ng/μl), 2 μl of 5× kinase buffer, 5 units of T4 polynucleotide kinase, and 3 μl of [γ-32P]ATP (3,000 Ci/mmol at 10 mCi/ml) incubated at 37°C for 1 h. The reaction was stopped by adding 90 μl of TE buffer (10 mM Tris-HCl pH 7.4 and 1 mM EDTA). To separate the labeled probe from the unbound ATP, the reaction mixture was eluted in a Nick column (Pharmacia) according to the manufacturer's instructions. Eight micrograms of crude nuclear proteins were incubated for 15 min on ice in binding buffer [10 mM Tris-HCl pH 8.0, 25 mM KCl, 0.5 mM DTT, 0.1 mM EDTA pH 8.0, 5% glycerol, 5 mg/ml BSA, 100 μg/ml tRNA, and 50 μg/ml poly(dI-dC)], in a final volume of 15 μl. Labeled probe (∼50,000 cpm) was added and the reaction was incubated for 20 min at room temperature. Where indicated, specific competitor oligonucleotide was added before the addition of labeled probe and incubated for 10 min on ice. For supershift assays, antibodies were added after incubation with labeled probe for a further 30 min at room temperature. Protein-DNA complexes were resolved by electrophoresis at 4°C on a 5% acrylamide gel and subjected to autoradiography. Antibodies against COUP-TF II and p65 were from Santa Cruz Biotechnology. Crude nuclear extracts (40 μg) from hearts were subjected to 10% SDS-polyacrylamide gel electrophoresis. Proteins were then transferred to Immobilon polyvinylidene fluoride transfer membranes (Millipore), and immunological detection was performed using a goat polyclonal antibody raised against COUP-TF II (dilution 1:1,000). Detection was achieved using the enhanced chemiluminiscence (ECL) detection system (Amersham). Blots were also incubated with a rabbit antibody against β-tubulin (dilution 1:5,000) (Boehringer Mannheim), used as a control of equal abundance of nuclear extracts in the samples. Size of detected proteins was estimated using protein molecular mass standards (Life Technologies). Results are expressed as means ± SD of four or five mice. Significant differences were established by Student's t-test using the computer program GraphPad Instat. First, we investigated the effect of the CPT-I inhibitor etomoxir on the ratio of heart weight to body weight of mice to assess cardiac hypertrophy. Etomoxir administration (100 mg/kg/day) to mice for 1 day did not cause cardiac hypertrophy. In contrast, 10 days of etomoxir treatment resulted in a significant increase in both the net heart weight (20%) and the ratio of heart weight to body weight (24%) compared with the control group, indicating the presence of cardiac hypertrophy (Table 1). On the other hand, we assessed the effects of etomoxir on plasma glucose levels. In the nonfasted normoglycemic mice used in this study, drug treatment did not modify glucose levels compared with the control group.TABLE 1Etomoxir effects on cardiac hypertrophy and plasma glucose levelsControl1 Day EtomoxirControl10 Days EtomoxirBody weight (g)32.6 ± 2.832.7 ± 3.139.9 ± 5.539.1 ± 1.6Heart weight (g)0.15 ± 0.0070.15 ± 0.0150.20 ± 0.020.24 ± 0.03aP < 0.05.Heart weight/body weight (mg/g)0.46 ± 0.040.45 ± 0.040.50 ± 0.060.62 ± 0.09aP < 0.05.Plasma glucose (mg/dl)——99 ± 893 ± 11Mice were treated for one or 10 days with either 0.5% carboximethyl cellulose (control group) or 100 mg/kg/day of etomoxir (dissolved in 0.5% carboximethyl cellulose). Values are means ± SD of five animals per group.a P < 0.05. Open table in a new tab Mice were treated for one or 10 days with either 0.5% carboximethyl cellulose (control group) or 100 mg/kg/day of etomoxir (dissolved in 0.5% carboximethyl cellulose). Values are means ± SD of five animals per group. We first examined the effects of etomoxir treatment for either 1 or 10 days on the mRNA levels of ACO and medium chain acyl-CoA (MCAD) genes. The former catalyzes the rate-limiting step of peroxisomal β-oxidation of fatty acids and its transcription is controlled by PPARα (4Desvergne B. Wahli W. Peroxisome proliferator-activated receptors: nuclear control of metabolism.Endocr. Rev. 1999; 20: 649-688Google Scholar). The second, MCAD, is also a PPARα-target gene that catalyses a rate limiting step in the mitochondrial β-oxidation of medium-chain fatty acyl-thioesters (4Desvergne B. Wahli W. Peroxisome proliferator-activated receptors: nuclear control of metabolism.Endocr. Rev. 1999; 20: 649-688Google Scholar). In the heart of 1-day etomoxir-treated mice, the mRNA levels of ACO were significantly increased (4.4-fold induction, P < 0.05) compared with control mice (Fig. 1A). Transcript levels of MCAD were not modified by this short treatment with etomoxir. These results are in agreement with previous studies showing that etomoxir (50 mg/kg body weight) administration for 1 day to mice caused a 4.5-fold induction in ACO mRNA levels in heart, whereas MCAD expression was not modified (15Djouadi F. Weinheimer C.J. Saffitz J.E. Pitchford C. Bastin J. Gonzalez F.J. Kelly D.P. A gender-related defect in lipid metabolism and glucose homeostasis in peroxisome proliferator- activated receptor alpha- deficient mice.J. Clin. Invest. 1998; 102: 1083-1091Google Scholar). Therefore, these results confirm the validity of our etomoxir treatment. Since inhibition by etomoxir of the transport of long-chain fatty acyl-CoA compounds into the mitochondria increases its accumulation in the cytoplasm, we also studied the effects of this drug on cytosolic acyl-CoA thioesterase (CTE). CTE catalyzes the hydrolysis of acyl-CoAs to free fatty acids and CoA, and as a consequence, it is an important mediator in cellular processes regulated by intracellular levels of nonesterified fatty acids and acyl-CoAs. In the heart of 1-day etomoxir-treated mice, the mRNA levels of CTE were 2-fold higher (P < 0.05) than in control mice. The changes in CTE mRNA after a single etomoxir administration are consistent with the reported regulation of this gene by PPARα in heart (16Hunt M.C. Lindquist P.J.G. Peters J.M. Gonzalez F.J. Diczfalusy U. Alexson S.E. Involvement of the peroxisome proliferator-activated receptor alpha in regulating long-chain acyl-CoA thioesterases.J. Lipid Res. 2000; 41: 814-823Google Scholar). When we examined the expression of ACO in the heart of 10-days etomoxir-treated mice, a dramatic reduction was observed in ACO mRNA (96% reduction, P < 0.05) (Fig. 1B) compared with control mice. In contrast, etomoxir treatment did not affect the mRNA expression of two genes of the mitochondrial β-oxidation system MCAD and muscle-type carnitine palmitoyl-transferase (M-CPT-I), being the latter a PPAR-target gene (17McGarry J.D. Brown N.F. The mitochondrial carnitine palmitoyltransferase system. From concept to molecular analysis.Eur. J. Biochem. 1997; 244: 1-14Google Scholar, 18Mascaró C. Acosta E. Ortiz J.A. Marrero P.F. Hegardt F.G. Haro D. Control of human muscle-type carnitine palmitoyltransferase I gene transcription by peroxisome proliferator-activated receptor.J. Biol. Chem. 1998; 273: 8560-8563Google Scholar) that catalyzes the entry of long-chain fatty acids into the mitochondrial matrix (18Mascaró C. Acosta E. Ortiz J.A. Marrero P.F. Hegardt F.G. Haro D. Control of human muscle-type carnitine palmitoyltransferase I gene transcription by peroxisome proliferator-activated receptor.J. Biol. Chem. 1998; 273: 8560-8563Google Scholar). Similarly, the expression of uncoupling protein 3 (UCP-3) and UCP-2, mitochondrial carriers localized in the inner mitochondrial membrane that have been implicated in fatty acid utilization and are regulated by PPARα (19Ricquier D. Bouillad F. The uncoupling protein homologues: UCP1, UCP2, UCP3, StUCP and AtUCP.Biochem. J. 2000; 345: 161-179Google Scholar), was not affected by the treatment. CTE mRNA levels were 2.3-fold higher in etomoxir-treated mice compared with the control group. This finding, in agreement with a previous report (16Hunt M.C. Lindquist P.J.G. Peters J.M. Gonzalez F.J. Diczfalusy U. Alexson S.E. Involvement of the peroxisome proliferator-activated receptor alpha in regulating long-chain acyl-CoA thioesterases.J. Lipid Res. 2000; 41: 814-823Google Scholar), suggests that CTE is also induced in a PPARα-independent manner, since this is the only PPARα-target gene that is up-regulated after 10 days of etomoxir treatment. In fact, it has been postulated that other lipid-activated nuclear receptors such as PPARδ or COUP-TF II, may regulate CTE in heart (16Hunt M.C. Lindquist P.J.G. Peters J.M. Gonzalez F.J. Diczfalusy U. Alexson S.E. Involvement of the peroxisome proliferator-activated receptor alpha in regulating long-chain acyl-CoA thioesterases.J. Lipid Res. 2000; 41: 814-823Google Scholar). Given the crucial role of PPARα in the control of cardiac lipid metabolism (20Djouadi F. Brandt J.M. Weinheimer C.J. Leone T.C. Gonzalez F.J. Kelly D.P. The role of the peroxisome proliferator-activated receptor alpha (PPAR alpha) in the control of cardiac lipid metabolism.Prostaglandins Leukot. Essent. Fatty Acids. 1999; 60: 339-343Google Scholar) and the previous results showing down-regulation of this transcription factor during the development of pressure overload-induced ventricular hypertrophy in mice (7Sack M.N. Disch D.L. Rockman H.A. Kelly D.P. A role for Sp and nuclear receptor transcription factors in a cardiac hypertrophy growth program.Proc. Natl. Acad. Sci. USA. 1997; 94: 6438-6443Google Scholar, 8Barger P.M. Brandt J.M. Leone T.C. Weinheimer C.J. Kelly D.P. Deactivation of peroxisome proliferator-activated receptor-a during cardiac hypertrophic growth.J. Clin. Invest. 2000; 105: 1723-1730Google Scholar), we finally studied whether the effects of etomoxir treatment in heart were mediated by reduced expression of this transcription factor. PPARα mRNA levels were not modified after 10 days of etomoxir treatment, indicating that changes in PPARα expression were not responsible for the effects of etomoxir. The induction of PPARα-target genes, such as ACO and MCAD, by PPARα activators is reduced in livers of RXRα-deficient mice (21Wan H-J.Y. Cai Y. Lungo W. Fu P. Locker J. French S. Sucov H.M. Peroxisome proliferator-activated receptor alpha-mediated pathways are altered in hepatocyte-specific retinoid X receptor alpha-deficient mice.J. Biol. Chem. 2000; 275: 28285-28290Google Scholar). Therefore, in order to study whether reduced availability of the PPARα heterodimeric partner RXR was responsible for the reduced transcriptional activity of the ACO gene in 10-days etomoxir-treated mice, we determined the transcript levels of RXRα and RXRγ. Etomoxir treatment for 10 days did not modify the mRNA expression of these transcription factors in heart compared with control mice (Fig. 2). In addition, we studied the mRNA expression of PPARγ coactivator 1 (PGC-1), which directly interacts with PPARα and has been postulated as a regulator of mitochondrial β-oxidation (22Vega R.B. Huss J.M. Kelly D.P. The coactivator PGC-1 cooperates with peroxisome proliferator-activated receptor alpha in transcriptional control of nuclear genes encoding mitochondrial fatty acid oxidation enzymes.Mol. Cell. Biol. 2000; 20: 1868-1876Google Scholar). PGC-1 mRNA was not altered in the heart of 10-days etomoxir-treated mice. All these findings make unlikely a role for RXR and PGC-1 in the changes observed after etomoxir treatment for 10 days. EMSAs were performed to examine the interaction of PPAR with its cis-regulatory element using a 32P-labeled PPRE probe and cardiac nuclear extracts from control and etomoxir-treated mice. The PPRE probe formed four complexes with cardiac nuclear proteins (complexes I to IV, Fig. 3A). The four complexes represented specific PPRE-protein interactions, since they were competed with a molar excess of unlabeled probe but not by an equivalent amount of a nonspecific competitor oligonucleotide (NSP). These results suggest that several endogenous cardiac nuclear proteins bind PPRE. In nuclear extracts from hearts of 1-day etomoxir-treated mice, no significant changes were observed in DNA binding activity to the PPRE probe compared with control animals (Fig. 3B). In contrast, despite the lack of induction in the transcriptional rate of PPARα-target genes after etomoxir treatment for 10 days, this drug increased th
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